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UNIVERSIDAD SAN FRANCISCO DE QUITO USFQ Carla Nicole UNIVERSIDAD SAN FRANCISCO DE QUITO USFQ Colegio de Ciencias Biológicas y Ambientales Detection of Anaplasma, Babesia and Mycoplasma in hunting dogs from the riverbank of Napo River. Carla Nicole Villamarín Uquillas Ingeniería en Procesos Biotecnológicos Trabajo de fin de carrera presentado como requisito para la obtención del título de Ingeniera en Procesos Biotecnológicos Quito, 4 de mayo de 2020 2 UNIVERSIDAD SAN FRANCISCO DE QUITO USFQ Colegio de Ciencias Biológicas y Ambientales HOJA DE CALIFICACIÓN DE TRABAJO DE FIN DE CARRERA Detection of Anaplasma, Babesia and Mycoplasma in hunting dogs from the riverbank of Napo River. Carla Nicole Villamarín Uquillas Nombre del profesor, Título académico Verónica Barragán, PhD Quito, 4 de mayo de 2020 3 DERECHOS DE AUTOR Por medio del presente documento certifico que he leído todas las Políticas y Manuales de la Universidad San Francisco de Quito USFQ, incluyendo la Política de Propiedad Intelectual USFQ, y estoy de acuerdo con su contenido, por lo que los derechos de propiedad intelectual del presente trabajo quedan sujetos a lo dispuesto en esas Políticas. Asimismo, autorizo a la USFQ para que realice la digitalización y publicación de este trabajo en el repositorio virtual, de conformidad a lo dispuesto en el Art. 144 de la Ley Orgánica de Educación Superior. Nombres y apellidos: Carla Nicole Villamarín Uquillas Código: 00132945 Cédula de identidad: 0931084776 Lugar y fecha: Quito, mayo de 2020 4 ACLARACIÓN PARA PUBLICACIÓN Nota: El presente trabajo, en su totalidad o cualquiera de sus partes, no debe ser considerado como una publicación, incluso a pesar de estar disponible sin restricciones a través de un repositorio institucional. Esta declaración se alinea con las prácticas y recomendaciones presentadas por el Committee on Publication Ethics COPE descritas por Barbour et al. (2017) Discussion document on best practice for issues around theses publishing, disponible en http://bit.ly/COPETheses. UNPUBLISHED DOCUMENT Note: The following capstone project is available through Universidad San Francisco de Quito USFQ institutional repository. Nonetheless, this project – in whole or in part – should not be considered a publication. This statement follows the recommendations presented by the Committee on Publication Ethics COPE described by Barbour et al. (2017) Discussion document on best practice for issues around theses publishing available on http://bit.ly/COPETheses. 5 RESUMEN La ribera del río Napo es una zona rica en vida silvestre, que además se comparte con las comunidades amazónicas y animales domésticos. En la Amazonía ecuatoriana, la caza sigue siendo un factor crítico en la subsistencia de las comunidades nativas y los perros domésticos se incorporan en esta actividad con fines de asistencia y apoyo. Se sabe que especies de hemoparásitos pueden ser alojadas en los eritrocitos de estos animales, sin embargo, la presencia de este grupo de microorganismos también se ha reportado en animales salvajes como ciervos, cánidos, felinos, suinos y roedores, lo que indica una posible zoonosis entre ambos grupos. Este estudio evalúa por primera vez la presencia de tres hemoparásitos con potencial zoonótico en 53 perros de caza (Canis lupus familiaris). Se utiliza a los perros de caza como animal “centinela” que nos permite comprender la circulación de enfermedades infecciosas en 5 comunidades locales de la ribera del río Napo, en la Amazonía ecuatoriana. La detección de los hemoparásitos se llevó a cabo amplificando el gen de ARN 16s por PCR convencional y PCR SYBR Green en tiempo real. Además, las especies de hemoparásitos detectadas fueron identificadas por secuenciación. En la detección de Babesia spp. y Anaplasma spp. se obtuvo resultados negativos para todas las muestras analizadas. Para Mycoplasma spp. se observó una positividad total de 30.18% (16/53) entre 4 de los 5 puntos de muestreo, pues una de las localidades no presento positivos. Se identificaron dos especies: M. haemocanis y Candidatus Mycoplasma haematoparvum. Los resultados indican que estos hemoparásitos están circulando en la zona y por lo tanto podrían estar infectando también la vida silvestre. Sin embargo, no es posible afirmar la direccionalidad de esta transmisión a partir de los resultados aquí presentados. Esta investigación corresponde a la primera etapa del análisis de los hemoparásitos que circulan entre animales domésticos y salvajes en la zona, y sus implicaciones y abre la puertas a nuevos estudios de investigación sobre los mecanismos de contagio y sus consecuencias sobre la biodiversidad. Así mismo, existe un gran potencial de tales estudios para ser utilizado con fines de conservación de vida silvestre. Palabras clave: Anaplasma, Babesia, Mycoplasma, Canis lupus familiaris, zoonosis, vida silvestre, hemoparásitos, ribera del rio Napo. 6 ABSTRACT The riverbank of the Napo River is a wildlife rich area, which is also shared with Amazonian communities and domestic animals. In the Ecuadorian Amazon, hunting is still a critical factor in native communities’ subsistence and domestic dogs are incorporated as assistance and support. It is known that species of hemoparasites can be hosted in the erythrocytes of these animals, however, the presence of this group of microorganisms has also been reported in wild animals such as deer, canids, felids, swine, and rodents, indicating a possible zoonosis between both groups. This study evaluates for the first time the presence of three hemoparasites with zoonotic potential in 53 hunting dogs (Canis lupus familiaris). Hunting dogs are used as "sentinel" animals, that allows to understand the circulation of infectious diseases in 5 local communities on the banks of the Napo River, in the Ecuadorian Amazon. The detection was carried out by amplifying the 16s RNA gene by conventional PCR and PCR SYBR Green in real time. In addition, the species of the parasites detected were identified by sequencing. In the detection of Babesia spp. and Anaplasma spp. negative results were obtained for all the samples analyzed. For Mycoplasma spp. An overall positivity of 30.18% (16/53) was observed among 4 of the 5 sampling spots, as one of the localities sampled did not show any positives. Two species were identified: M. haemocanis and Candidatus Mycoplasma haematoparvum. The results indicate that these hemoparasites are circulating in the area and therefore could be infecting wildlife as well. However, it is not possible to affirm the directionality of this transmission from the results presented here. This research corresponds to the first stage of the analysis of the hemoparasites circulating between domestic and wild animals in the area, and its implications. It opens the doors to new research studies about mechanisms of contagion and its consequences on biodiversity. Likewise, there is a great potential of such studies to be used for wildlife conservation purposes. Key words: Anaplasma, Babesia, Mycoplasma, Canis lupus familiaris, subsistence hunting, zoonoses, wild fauna, hemoparasites, riverbank of Napo River. 7 TABLE OF CONTENTS Resumen .......................................................................................................................................... 5 Abstract ........................................................................................................................................... 6 Introduction ................................................................................................................................... 10 Materials and methods .................................................................................................................. 14 Sample collection ...................................................................................................................... 14 DNA extraction ......................................................................................................................... 14 Hemoparasite detection ............................................................................................................. 15 Sequencing and taxonomic assignation..................................................................................... 16 Results ........................................................................................................................................... 17 No PCR inhibiting compounds were found in DNA extracts ................................................... 17 Hemoparasites found in dog blood samples .............................................................................. 17 Discussion ..................................................................................................................................... 18 Data indicates absence of Anaplasma spp. and Babesia spp. ................................................... 18 Detection assays revealed occurrence of Mycoplasma spp. ..................................................... 21 Conclusions ................................................................................................................................... 25 References ..................................................................................................................................... 26 Tables ............................................................................................................................................ 31 Figures........................................................................................................................................... 33 Apendix
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